The goal of this application is to identify the molecular and cellular mechanisms that control the ability of lymph node resident lymphatic endothelial cells to induce peripheral immunological tolerance. We and two other groups have described a mechanism in which stromal cells in lymph nodes directly express proteins that are normally associated with other tissues. Direct presentation of epitopes derived from these proteins to CD8 T cells leads to abortive proliferation and deletion. Populations of lymph node stromal cells that have been shown to mediate this process include: extra thymic Aire-expressing cells (eTAC), fibroblastic reticular cells, and lymphatic endothelial cells. In particular, we have shown that CD8 T cell tolerance to an epitope derived from tyrosinase, a protein whose expression is normally confined to melanocytes and retinal pigment epithelial cells, is mediated by lymph node resident lymphatic endothelial cells that adventitiously express it. The 3 lymph node stromal populations defined above express at least partially distinct subsets of peripheral tissue antigens. Peripheral tissue antigens are also expressed in the thymus under the influence of the autoimmune regulatory element Aire. Aire also controls the expression of peripheral tissue antigens in the eTAC lymph node stromal subset. However, neither fibroblastic reticular cells nor lymphatic endothelial cells express Aire, and the mechanisms that control transcription of peripheral tissue antigen mRNAs in these lymph node stromal populations are unknown. One goal of this application is to identify transcription factors that lead to peripheral tissue antige expression in lymphatic endothelial cells. This will illuminate new mechanisms for control of peripheral tissue antigen expression, apart from Aire, that underlie the development of systemic peripheral tolerance. Our preliminary work has established that the lymph node resident lymphatic endothelial cells differ from their counterparts that form tissue lymphatic vessels by elevated expression of peripheral tissue antigens and the ability to present tyrosinase epitopes to tyrosinase-specific T cells. Lymph node resident lymphatic endothelial cells also express a number of immunologically relevant molecules that are not expressed by their tissue lymphatic counterparts. Of these, the most important is PD-L1, which we have shown to be responsible for T cell deletion during tolerance induction. We have also established the existence of subpopulations of lymphatic endothelial cells in the lymph node itself, and shown that tolerance to tyrosinase is primarily induced by those that form the medullary sinus. Thus, the ability of lymphatic endothelial cells to induce peripheral tolerance is regulated by the LN microenvironment. A second major goal of this application is to identify the cellular and molecular aspects of the LN microenvironment that regulate this tolerogenic phenotype. This work will set the stage for further understanding of the role lymphatic endothelial cells may play in self-tolerance and the development of autoimmune disease in humans.